Figure 3

Aβ induced BMAL1 and CBP protein degradation in the nucleus of HT22 cells . (A-D) Aβ-induced BMAL1 and CBP protein degradation was measured in the nucleus of HT22 cells. (A) HT22 cells were incubated with vehicle or Aβ (2 μM) for CT12, 24 after cell synchronization. Western blot analysis showed that BMAL1 and CBP protein expression was decreased in the nucleus at CT24. (B) Densitometric analysis of BMAL1 and CBP protein expression from three independent experiments. The intensities of the bands from BMAL1 and CBP proteins were normalized to Lamin B. Data are represented as mean ± SEM. ^** P < 0.01, ^# P < 0.05. (C) Photomicrograph of immunostaining with BMAL1 and CBP proteins in HT22 cells clearly shows that BMAL1 and CBP immunofluorescence are significantly decreased within the nucleus following treatment with 2 μM of Aβ for 24 h. (D) Densitometric analysis of BMAL1 and CBP immunofluorescence from three independent experiments. Data are represented as mean ± SEM. ^** P < 0.01, ^*** P < 0.001. (E) Aβ inhibited the formation of BMAL1-CBP BiFC complexes. Cos7 cells were transiently transfected with VC-BMAL1 and VN-CBP. 12 h post-transfection with VN-CBP and VC-BMAL1, cells were serum starved for 12 h and incubated with vehicle or Aβ for 24 h after cell synchronization. (F) Densitometric analysis of BMAL1-CBP BiFC complexes from three independent experiments. Data are represented as mean ± SEM. ^*** P < 0.001.